Bone regeneration agent including gelatin

ABSTRACT

It is an object of the present invention to provide a bone regeneration agent and a bone supplementation formulation, in which a supplementation material itself is capable of promoting bone regeneration. The present invention provides a bone regeneration agent which comprises a gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is Divisional application of U.S. patent applicationSer. No. 13/394,263, filed Mar. 5, 2012, which is a National Stage ofInternational Application No. PCT/JP2010/065108 filed Sep. 3, 2010,claiming priority based on Japanese Patent Application Nos. 2009-204218filed Sep. 4, 2009, and 2010-196678 filed Sep. 2, 2010, the contents ofall of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a bone regeneration agent and a bonesupplementation formulation, both of which comprise gelatin.

BACKGROUND ART

Body tissues are mainly composed of cells and extracellular matrixes(polymer structures). Extracellular matrixes which are secreted fromcells provide hydrated spaces in which cells act or cell scaffoldings.In addition, extracellular matrixes function as storage houses forvarious growth factors secreted from cells, and have a profoundinfluence on expression of cell functions or cell differentiation.Complicated interaction between extracellular matrixes and cells in aliving body has an influence on various vital activities.

In recent years, highly advanced medical technology, whereby severediseases are treated using artificial organs, gene therapy andregenerative medicine, has achieved remarkable success at clinicalsites, and it has attracted global attention. Among others, regenerativemedicine, whereby damaged tissues or organs are regenerated using threemain elements, namely, cells, growth factors and extracellular matrixesthat play main roles in tissue regeneration, has drawn attention as atreatment method for future generation. As a matter of fact, suchregenerative medicine has already been realized for cultured skins orcorneas.

Bone regeneration in the field of orthopedics or dentistry has attracteda great attention as an example of application of regenerative medicine.That is to say, when bone disease is developed in legs or lumbar parts,it causes abasia. When such bone disease is developed in tooth sections,it makes dietary intake difficult. Thus, bone disease is considered tocause a significant decrease in QOL. At present, representative examplesof a known bone regeneration therapeutic formulation include: Infuse (acombination of BMP-2 with a collagen sponge) for treating spinalinjuries; and BioOss (deproteinized, crushed cow bones), Puros (crushedhuman bone products), Gem21 (PDGF and βTCP), Osferion (βTCP) andTeruplug (Name used in the U.S.A.: FOUNDATION; a collagen sponge) usedas bone supplementation agents for regenerating alveolar bones.Properties known to be necessary for bone regeneration therapeuticformulations include: 1. strength for maintaining structures; 2. thesecurement of spaces for bone regeneration; 3. cell scaffoldings forbone regeneration; 4. differentiation and growth of cells necessary forbone regeneration; and 5. degradability attended with bone regeneration.

In the dental field, representative diseases, for which theabove-mentioned bone regeneration therapeutic formulations are known tobe used, include: 1. ridge augmentation; 2. socket preservation; 3.periodontal bone defect regeneration; 4. implantable bone regeneration;and 5. sinus lift. The importance of scaffolding materials for promotingbone regeneration in the aforementioned diseases has been recognized.However, under the current circumstances, the aforementionedformulations rely on the bone regeneration ability of an agent (BMP orRDGF) contained therein. Such scaffolding materials have been believedto function as products for ensuring a space (strength) in an affectedarea. In many cases, inorganic materials have been widely used as suchscaffolding materials.

In general, a collagen or a gelatin which is a denatured product of thecollagen has been widely used as a scaffolding material (organicmaterial) in regenerative medicine in many cases. It has been known thata highly oriented collagen plays an important role as a boneregeneration material in calcification (Non Patent Document 1). Thus, asponge made of collagen has been placed on the market as a boneregeneration material (Teruplug (FOUDATION; Olympus Terumo BiomaterialsCorp.)). However, a bone supplementation material using a collagensponge provides only a scaffolding for tissue formation, and a singleuse of such a collagen sponge is not able to form a bone (Non PatentDocument 2). Moreover, according to previous findings, a gelatin whichis a denatured product of collagen has never been known to have a boneregeneration effect. Regarding gelatin, it is described that 1. a singleuse of a gelatin sponge inhibits bone regeneration (Non Patent Document3), and that 2. a gelatin has bone regeneration ability that is lowerthan that of a collagen (Non Patent Document 4). Furthermore, it hasbeen reported that no bones are formed in R-TCP that is an artificialbone ingredient, as with a gelatin sponge prepared by performing a heattreatment on a collagen (Non Patent Documents 2 and 5). As describedabove, it has been recognized that a gelatin that has been widely usedas a base material in regenerative medicine is not suitable as ascaffolding material for bone regeneration therapeutic agents. Withregard to a supplementation material that cannot form a bone by itself,a method for impregnating the supplementation material with aplatelet-derived growth factor (PDGF) or a bone morphogenetic protein(BMP) which is a physiologically active substance has been studied(Patent Document 1). However, since a purified protein of suchphysiologically active substance is expensive, it has not yet beenwidely used.

On the other hand, biological polymers such as a gelatin have beenbroadly used as medical materials. As a result of the advancement ofgenetic engineering technology in recent years, a protein has beensynthesized by introducing a gene into Escherichia coli or yeast. Bythis method, various types of recombinant collagen-like proteins havebeen synthesized (for example, Patent Documents 2 and 3). Suchrecombinant collagen-like proteins are more advantageous than naturalgelatins in that they are excellent in terms of non-infectivity anduniformity, and in that since their sequences have been determined, thestrength and degradability thereof can be precisely designed. However,the intended use of the recombinant gelatin, which has been proposed sofar, is only the use as an alternative material of a natural gelatin.Naturally, the intended use of the recombinant gelatin as a boneregeneration agent has not been known.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] JP Patent Publication (Kokai) No. 2004-203829-   [Patent Document 2] U.S. Pat. No. 6,992,172-   [Patent Document 3] WO 2008/103041

Non Patent Documents

-   [Non Patent Document 1] Tabata et al., Biomaterials 19, 807-815,    1998.-   [Non Patent Document 2] Biomaterials 26: 2501-2507, 2005-   [Non Patent Document 3] Tabata et al., Journal of Neurosurgery 91,    851-856, 1999-   [Non Patent Document 4] Ishii et al., Shika-i Tenbou (Prospects for    Dentists), 97(3), 665-677, 2001-   [Non Patent Document 5] J Neurosurg 91: 851-856, 1999

SUMMARY OF INVENTION Object to be Solved by the Invention

It is an object to be solved by the present invention to provide a boneregeneration agent and a bone supplementation formulation, in which asupplementation material itself is capable of promoting boneregeneration.

Means for Solving the Object

As a result of intensive studies directed towards achieving theaforementioned object, the present inventors have found that a gelatinhaving an amino acid sequence derived from a partial amino acid sequenceof collagen, and more preferably a recombinant gelatin has an action toregenerate bone, thereby completing the present invention.

Thus, the present invention provides a bone regeneration agent whichcomprises a gelatin having an amino acid sequence derived from a partialamino acid of collagen.

Preferably, the gelatin having an amino acid sequence derived from apartial amino acid of collagen is a recombinant gelatin.

Preferably, the gelatin comprises repeats of a sequence represented byGly-X-Y characteristic to collagen and has a molecular weight of 2 KDato 100 KDa. wherein X and Y each independently represent an amino acidand a plurality of Gly-X-Y sequences may be the same or different.

Preferably, the gelatin comprises repeats of a sequence represented byGly-X-Y characteristic to collagen and has a molecular weight of 10 KDato 90 KDa. wherein X and Y each independently represent an amino acidand a plurality of Gly-X-Y sequences may be the same or different.

Preferably, the gelatin comprises repeats of a sequence represented byGly-X-Y characteristic to collagen and has two or more sequences of celladhesion signals in a single molecule wherein X and Y each independentlyrepresent an amino acid and a plurality of Gly-X-Y sequences may be thesame or different.

Preferably, the cell adhesion signal sequence is an amino acid sequencerepresented by Arg-Gly-Asp.

Preferably, the amino acid sequence of the gelatin does not comprise anyof serine and threonine.

Preferably, the amino acid sequence of the gelatin does not comprise anyof serine, threonine, asparagine, tyrosine, and cysteine.

Preferably, the amino acid sequence of the gelatin does not comprise anamino acid sequence represented by Asp-Arg-Gly-Asp (SEQ ID NO: 2).

Preferably, the gelatin is represented by the following formula:

A-[(Gly-X-Y)_(n)]_(m)-B

wherein A represents any amino acid or amino acid sequence, B representsany amino acid or amino acid sequence, there exist n amino acids eachindependently represented by X, there exist n amino acids eachindependently represented by Y, n represents an integer from 3 to 100, mrepresents an integer of 2 to 10, and n Gly-X-Y sequences may be thesame or different.

Preferably, the gelatin is represented by the following formula:

(SEQ ID NO: 12) Gly-Ala-Pro-[(Gly-X-Y)₆₃]₃-Glywherein there exist 63 amino acids each independently represented by X,there exist 63 amino acids each independently represented by Y, and 63Gly-X-Y sequences may be the same or different.

Preferably, the gelatin has the following (1) or (2):

(1) the amino acid sequence shown in SEQ ID NO: 1; or(2) an amino acid sequence having 80% or more homology to the amino acidsequence shown in SEQ ID NO: 1 and having an action to regenerate bone.

Preferably, the gelatin is crosslinked.

Preferably, the crosslinking is carried out using an aldehyde,condensing agent or enzyme.

The present invention further provides a bone supplementationformulation which comprises the aforementioned bone regeneration agentof the present invention.

The present invention further provides a method for inducing boneregeneration which comprises administering a gelatin having an aminoacid sequence derived from a partial amino acid of collagen to a subjectin need of bone regeneration.

The present invention further provides a use of a gelatin having anamino acid sequence derived from a partial amino acid of collagen, forthe production of a bone regeneration agent or a bone supplementationformulation.

Effect of the Invention

The bone regeneration agent of the present invention is capable ofexhibiting an excellent bone regeneration effect by a supplementationmaterial itself, without using a physiologically active substance suchas a platelet-derived growth factor or a bone morphogenetic protein.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a HE stained image of an untreated rat calvarialbone-deficient model.

FIG. 2 is a HE stained image showing that recombinant gelatin powderswere embedded in a defect portion and a bone was then formed in theaffected area.

FIG. 3 is a HE stained image showing that pig-derived gelatin powderswere embedded in a defect portion.

FIG. 4 is a HE stained image showing that collagen powders were embeddedin a defect portion.

FIG. 5 is a HE stained image showing that a bovine cancellous boneBio-Oss (trade name), Osteohealth) which is an insoluble substrate wasembedded in a defect portion.

FIG. 6 shows electron micrographs of the insides of an R-Gel formulationand Teruplug.

FIG. 7 shows rat cranial bone-deficient models. Left: a macroscopicphotograph. Right: a μCT photograph (Micro-CT image: Tissue Eng (2007)13(3): 501-12).

FIG. 8 shows a method for calculating a bone regeneration percentage(analyzed from pathological specimens).

FIG. 9 shows a change over time in the bone regeneration percentage in arat cranial bone-deficient portion. R-Gel and an animal gelatinexhibited bone regeneration percentages higher than those of Bio-OssCancellous and Teruplug.

FIG. 10 shows pathological specimen photographs (H & E stained) takenone month and two months after transplantation of R-Gel and an animalgelatin into rat cranial bones. R-Gel (upper photographs) and the animalgelatin (lower photographs). One month after the transplantation, boneswere regenerated on the surfaces of the R-Gel and the animal gelatin.Two months after the transplantation, bones were regenerated at sitesaround the R-Gel, in which the R-Gel had been decomposed by cells, andbones with a few voids were regenerated. On the other hand, around theanimal gelatin, soft fibrous tissues were observed at sites in which theanimal gelatin had been decomposed by cells, and a large number ofcicatricial fibrous tissues were contained in the regenerated bones.

FIG. 11 shows macroscopic photographs of tooth extraction sites of dogmandibular premolar extraction models, which were taken immediatelyafter and two months after transplantation of infills.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereinafter the embodiments of the present invention will be describedin detail.

In the present invention, a gelatin having an amino acid sequencederived from a partial amino acid sequence of collagen is used as a boneregeneration agent. The bone regeneration agent of the present inventionis characterized in that a gelatin having an amino acid sequence derivedfrom a partial amino acid sequence of collagen exhibits boneregeneration action by itself. Accordingly, it is not necessary for thebone regeneration agent of the present invention to comprise othersubstances having bone regeneration action, other than the gelatinhaving an amino acid sequence derived from a partial amino acid sequenceof collagen. Thus, the bone regeneration agent of the present inventionpreferably does not comprise other substances having bone regenerationaction, other than the above-described gelatin. Specifically, the boneregeneration agent of the present invention preferably consists of thegelatin having an amino acid sequence derived from a partial amino acidsequence of collagen. The type of the collagen is not particularlylimited, as long as it is present in the nature. Preferred examples ofthe collagen include collagen types I, II, III, IV and V. More preferredexamples of the collagen include collagen types I, II and III. Inanother embodiment, preferred origins of the collagen are preferably ahuman, a bovine, a pig, a mouse and a rat. It is more preferably ahuman.

The gelatin used herein is not particularly limited, so long as thegelatin has an amino acid sequence derived from a partial amino acid ofcollagen. Preferably, the gelatin contains Gly-X-Y characteristic tocollagen. When gelatin/collagen is compared with other proteins in termsof the amino acid composition or sequence, the GXY sequence ischaracteristic to collagen and forms a highly specific partialstructure. Glycine accounts for approximately one-third of the partialstructure as a whole. Glycine is repeatedly found in the amino acidsequence at a rate of 1 out of every 3 amino acids. Glycine is thesimplest amino acid. There are few restrictions to arrangement of themolecular chain of glycine and thus glycine highly contributes toregeneration of the helix structure upon gelatinization. An amino acidrepresented by X or Y is rich in imino acid (proline or oxyproline) andthe imino acid accounts for 10% to 45% of the amino acid sequence as awhole.

The gelatin used in the present invention may be a gelatin which isderived from natural animal or may be a recombinant gelatin. When agelatin which is derived from natural animal is used, the origin of thegelatin is not particularly limited. The origin may be any animal suchas fish, bovine, pig or goat.

Among the above, it is preferred that the gelatin having an amino acidsequence derived from a partial amino acid of collagen is a recombinantgelatin. Since the recombinant gelatin is not derived from an animal butis an artificial gelatin, it is highly biocompatible supplementationmaterial where exogenous infection is avoided.

As a recombinant gelatin that can be used in the present invention, arecombinant gelatin having an amino acid sequence derived from a partialamino acid sequence of collagen can be used. Examples of a recombinantgelatin that can be used include, but are not limited to, recombinantgelatins described in EP1014176A2, U.S. Pat. No. 6,992,172,WO2004-85473, and WO2008/103041. A recombinant gelatin preferably usedas the recombinant gelatin of the present invention is described below.

The recombinant gelatin used in the present invention has originalproperties of naturally occurring gelatin and thus it is highlybiocompatible. In addition, the recombinant gelatin is not directlyobtained from natural sources and thus has no risk of causing BSE or thelike. In this regard, it has an excellent property of beingnon-infectious. In addition, the recombinant gelatin used in the presentinvention is more homogenous than naturally occurring gelatin. Further,the recombinant gelatin has a predetermined sequence. Thus, it ispossible to precisely design the recombinant gelatin in terms ofstrength and degradability with few errors by crosslinking or the likedescribed below.

The molecular weight of the recombinant gelatin used in the presentinvention is preferably 2 KDa to 100 KDa, more preferably 2.5 KDa to 95KDa, further preferably 5 KDa to 90 KDa, and most preferably 10 KDa to90 KDa.

Preferably, the recombinant gelatin used in the present inventioncontains repeats of a sequence represented by Gly-X-Y characteristic tocollagen. Here, a plurality of sequences each represented by Gly-X-Y maybe the same or different. Gly in Gly-X-Y represents glycine. X and Y inGly-X-Y represent any amino acids (and preferably any amino acids otherthan glycine). When gelatin/collagen is compared with other proteins interms of the amino acid composition or sequence, the GXY sequence ischaracteristic to collagen and forms a highly specific partialstructure. Glycine accounts for approximately one-third of the partialstructure as a whole. Glycine is repeatedly found in the amino acidsequence at a rate of 1 out of every 3 amino acids. Glycine is thesimplest amino acid. There are few restrictions to arrangement of themolecular chain of glycine and thus glycine highly contributes toregeneration of the helix structure upon gelatinization. Preferably, anamino acid represented by X or Y is rich in imino acid (proline oroxyproline) and the imino acid accounts for 10% to 45% of the amino acidsequence as a whole. Amino acids forming the GXY repeat structureaccount for preferably 80% or more, more preferably 95% or more, andmost preferably 99% or more of the amino acid sequence as a whole.

A generally available gelatin contains charged polar amino acids anduncharged polar amino acids at a ratio of 1:1. Here, the term “polaramino acid” specifically refers to cysteine, aspartic acid, glutamicacid, histidine, lysine, asparagine, glutamine, serine, threonine,tyrosine, or arginine. In particular, the term “uncharged polar aminoacid” refers to cysteine, asparagine, glutamine, serine, threonine, ortyrosine. The percentage of polar amino acids relative to all aminoacids constituting the recombinant gelatin used in the present inventionis 10% to 40% and preferably 20% to 30%. In addition, the percentage ofuncharged polar amino acids relative to the polar amino acids ispreferably 5% to less than 20% and more preferably less than 10%.Further, the amino acid sequence does not contain one amino acid andpreferably two amino acids or more selected from among serine,threonine, asparagine, tyrosine, and cysteine.

In general, it is known that a polypeptide contains a minimal amino acidsequence that functions as a cell adhesion signal sequence (e.g.,“Pathophysiology” (Byotai Seiri) Vol. 9, No. 7 (1990), p. 527, NagaiShoten Co., Ltd.). It is preferable for a single molecule of therecombinant gelatin used in the present invention to have at least twocell adhesion signal sequences. Specifically, amino acids are shown byone-letter notation in a cell adhesion signal sequence. In view of anincrease in types of adhering cells, examples of such sequence are:preferably an RGD sequence, an LDV sequence, an REDV (SEQ ID NO: 3)sequence, a YIGSR (SEQ ID NO: 4) sequence, a PDSGR (SEQ ID NO: 5)sequence, an RYVVLPR (SEQ ID NO: 6) sequence, an LGTIPG (SEQ ID NO: 7)sequence, an RNIAEIIKDI (SEQ ID NO: 8) sequence, an IKVAV (SEQ ID NO: 9)sequence, an LRE sequence, a DGEA (SEQ ID NO: 10) sequence, and an HAVsequence, more preferably an RGD sequence, a YIGSR (SEQ ID NO: 4)sequence, a PDSGR (SEQ ID NO: 5) sequence, an LGTIPG (SEQ ID NO: 7)sequence, an IKVAV (SEQ ID NO: 9) sequence, and an HAV sequence; andparticularly preferably an RGD sequence. Among the RGD sequence, an ERGD(SEQ ID NO: 11) sequence is preferred.

In terms of arrangement of RGD sequences in the recombinant gelatin usedin the present invention, the number of amino acids present between twoRGD sequences is preferably 0 to 100 and more preferably 25 to 60.Preferably, the number of amino acids is not uniformly determined.

In view of cell adhesion/growth, the number of such minimal amino acidsequences in a single protein molecule is preferably 3 to 50, morepreferably 4 to 30, particularly preferably 5 to 20, and most preferably12.

The percentage of RGD motifs in the recombinant gelatin used in thepresent invention related to the total number of amino acids ispreferably at least 0.4%. If the recombinant gelatin comprises 350 aminoacids or more, each stretch of 350 amino acids contains preferably atleast one RGD motif. The percentage of RGD motifs related to the totalnumber of amino acids is more preferably at least 0.6%, furtherpreferably at least 0.8%, still further preferably at least 1.0%, evenfurther preferably at least 1.2%, and most preferably at least 1.5%. Thenumber of RGD motifs in the recombinant gelatin is preferably at least4, more preferably 6, further preferably 8, and even further preferably12 to 16 per 250 amino acids. A percentage of RGD motifs of 0.4%corresponds to at least one RGD sequence per 250 amino acids. The numberof RGD motifs is represented by an integer. Therefore, in order toachieve a percentage of RGD motifs of 0.4%, it is necessary for agelatin comprising 251 amino acids to contain at least two RGDsequences. Preferably, the recombinant gelatin of the present inventioncontains at least 2 RGD sequences per 250 amino acids, more preferablyat least 3 RGD sequences per 250 amino acids, and further preferably atleast 4 RGD sequences per 250 amino acids. In another embodiment, therecombinant gelatin of the present invention comprises at least 4,preferably 6, more preferably 8, and further preferably 12 to 16 RGDmotifs.

In addition, the recombinant gelatin may be partially hydrolyzed.

Preferably, the recombinant gelatin used in the present invention has astructure comprising repeats of A-[(Gly-X-Y)n]m-B. Here, “m” is aninteger of preferably 2 to 10 and more preferably 3 to 5. In addition,“n” is an integer of preferably 3 to 100, more preferably 15 to 70, andmost preferably 50 to 65.

Preferably, a plurality of naturally occurring collagen sequence unitsare bound to form a repeat unit. The term “naturally occurring collagen”used herein may refer to any naturally occurring collagen. However,preferable examples thereof include type-I, type-II, type-III, type-IV,and type-V collagens. More preferably, type-I, type-II, and type-IIIcollagens are used. In another embodiment, the origin of such collagenis preferably a human, bovine, pig, mouse, or rat and it is morepreferably a human.

The isoelectric point of the recombinant gelatin used in the presentinvention is preferably 5 to 10, more preferably 6 to 10, and furtherpreferably 7 to 9.5.

Preferably, the recombinant gelatin is not deaminated.

Preferably, the recombinant gelatin is not procollagen or does notcomprise procollagen.

Preferably, the recombinant gelatin does not comprise telopeptide.

Preferably, the recombinant gelatin is a substantially pure collagenmaterial prepared from a nucleic acid encoding a naturally occurringcollagen.

Particularly preferably, the recombinant gelatin used in the presentinvention is a recombinant gelatin having the following (1) or (2):

(1) the amino acid sequence shown in SEQ ID NO: 1; or

(2) an amino acid sequence having 80% or more, more preferably 90% ormore, and most preferably 95% or more homology to the amino acidsequence shown in SEQ ID NO: 1, and having an action to regenerate bone.

The recombinant gelatin used in the present invention can be produced bya gene recombination technique known to persons skilled in the art. Forinstance, it can be produced according to the method described inEP1014176A2, U.S. Pat. No. 6,992,172, WO2004/85473, or WO2008/103041.Specifically, a transformant is produced by obtaining a gene encodingthe amino acid sequence of a predetermined recombinant gelatin,incorporating the gene into an expression vector to prepare arecombinant expression vector, and introducing the vector into anappropriate host. The obtained transformant is cultured in anappropriate medium to produce a recombinant gelatin. Therefore, therecombinant gelatin used in the present invention can be prepared bycollecting the produced recombinant gelatin from the culture product.

The recombinant gelatin used in the present invention can be chemicallymodified depending on the application thereof. Chemical modification maybe performed via introduction of a low molecular compound or a differentpolymer (e.g., a biopolymer (sugar or protein), a synthetic polymer, orpolyamide) into a carboxyl group or an amino group of a side chain ofthe recombinant gelatin or crosslinking between recombinant gelatinchains. For example, a carbodiimide-based condensing agent is used forintroduction of a low molecular compound into the gelatin.

The crosslinking agent used in the present invention is not particularlylimited, as long as the present invention can be carried out. It may bea chemical crosslinking agent or an enzyme. Examples of a chemicalcrosslinking agent include formaldehyde, glutaraldehyde, carbodiimide,and cyanamide. Preferably, formaldehyde or glutaraldehyde is used.Further, crosslinking of a gelatin can be conducted by light irradiationto a gelatin into which a photoreactive group has been introduced, lightirradiation under the presence of a photosensitizer, or the like.Examples of a photoreactive group include a cinnamyl group, a coumaringroup, a dithiocarbamyl group, xanthene dye, and camphorquinone. Amongthe aforementioned crosslinking agent, glutaraldehyde is most preferred.

In a case in which enzymatic crosslinking is carried out, an enzyme usedis not particularly limited, as long as it has an action of causingcrosslinking between gelatin chains. However, crosslinking can becarried out using preferably transglutaminase or laccase and mostpreferably transglutaminase. Examples of proteins that are enzymaticallycrosslinked by transglutaminase include, but are not particularlylimited to, proteins having lysine residues and glutamine residues. Amammalian-derived or microorganism-derived transglutaminase may be used.Specific examples thereof include: the Activa series (produced byAjinomoto Co., Inc.); commercially available mammalian-derivedtransglutaminases serving as reagents such as guinea pig liver-derivedtransglutaminase, goat-derived transglutaminase, and rabbit-derivedtransglutaminase (produced by Oriental Yeast Co., Ltd., Upstate USAInc., Biodesign International, etc.); and a human-derived bloodcoagulation factor (Factor XIIIa, Haematologic Technologies, Inc.).

Crosslinking of the gelatin comprises the following two steps: a step ofmixing a gelatin solution and a crosslinking agent; and a step ofcausing a reaction in the obtained homogenous solution.

According to the present invention, the mixing temperature for treatingthe gelatin with a crosslinking agent is not particularly limited, aslong as the solution can be homogenously agitated. However, it ispreferably 0° C. to 40° C., more preferably 0° C. to 30° C., furtherpreferably 3° C. to 25° C., still further preferably 3° C. to 15° C.,even further preferably 3° C. to 10° C., and particularly preferably 3°C. to 7° C.

After agitation of the gelatin and the crosslinking agent, thetemperature can be increased. The reaction temperature is notparticularly limited, as long as crosslinking can proceed. However, inview of denaturation or degradation of the gelatin, it is substantially0° C. to 60° C., preferably 0° C. to 40° C., more preferably 3° C. to25° C., further preferably 3° C. to 15° C., still further preferably 3°C. to 10° C., and particularly preferably 3° C. to 7° C.

In the present invention, the above-described gelatin having an aminoacid sequence derived from a partial amino acid sequence of collagen isadministered to a subject in need of bone regeneration (for example, amammal such as a human), so that bone regeneration can be induced.

The bone regeneration agent of the present invention can be used as abone supplementation formulation.

The dose, usage and dosage form of the bone regeneration agent and thebone supplementation formulation of the present invention can bedetermined, as appropriate, depending on the intended use thereof. Forexample, the bone regeneration agent of the present invention may bedirectly administered to a target site in a living body. Otherwise, thebone regeneration agent of the present invention may be suspended in aliquid excipient including an aqueous solvent such as a distilled waterfor injection, a normal saline for injection, or a buffer with pH 5 to 8(a phosphate buffer, a citrate buffer, etc.), and it may be thenadministered by injection, coating, etc. Moreover, the bone regenerationagent of the present invention may be mixed with a suitable excipient soas to prepare an ointment, a gel, a cream, etc., and it may be thenapplied. That it to say, the administration form of the boneregeneration agent of the present invention may be either oraladministration or parenteral administration (for example, intravenousadministration, intramuscular administration, subcutaneousadministration, intracutaneous administration, etc.).

Examples of the dosage form of the bone regeneration agent of thepresent invention include: oral administration agents such as a tablet,a powdery agent, a capsule, a granular agent, an extract or a syrup; andparenteral administration agents such as an injection (for example,intravenous injection, intramuscular injection, subcutaneous injection,intracutaneous injection, etc.).

Preferably, the bone regeneration agent and the bone supplementationformulation of the present invention can be directly administered to abone defect site in a living body. Thus, when the bone regenerationagent of the present invention is locally administered, its form is notparticularly specified. Examples of the form include a sponge, a film, anon-woven fabric, a fiber (tube), a particle, and a mesh.

The bone regeneration agent and the bone supplementation formulation ofthe present invention can be prepared according to a method known to aperson skilled in the art. For example, when the pharmaceutical carrieris a liquid, the bone regeneration agent and the bone supplementationformulation of the present invention can be dissolved or dispersed inthe liquid. On the other hand, when the pharmaceutical carrier is apowder, the bone regeneration agent and the bone supplementationformulation of the present invention can be mixed with or adsorbed onthe powders. Furthermore, the bone regeneration agent and the bonesupplementation formulation of the present invention may also comprisepharmaceutically acceptable additives (for example, a preservative, astabilizer, an antioxidant, an excipient, a binder, a disintegrator, awetting agent, a lubricant, a coloring agent, an aromatic agent, acorrigent, a coating, a suspending agent, an emulsifier, a solubilizer,a buffer, an isotonizing agent, a plasticizer, a surfactant, a soothingagent, etc.), as necessary.

The dosage of the gelatin is not particularly limited. For example, itis 1 to 100 mg, and preferably 1 to 50 mg per cm² of surface area of aliving body, to which the gelatin is administered.

Examples of a target disease, to which the bone regeneration agent andthe bone supplementation formulation of the present invention isadministered, include bone defect caused by external injury, oralsurgery disease, osteoporosis, and arthropathy.

The present invention will be more specifically described in thefollowing examples. However, these examples are not intended to limitthe scope of the present invention.

EXAMPLES

As a recombinant gelatin, CBE3 (WO2008-103041) described below wasprepared.

CBE3

Molecular weight: 51.6 kD

Structure: Gly-Ala-Pro[(Gly-X-Y)₆₃]₃Gly (SEQ ID NO: 12)

Number of amino acids: 571Number of RGD sequences: 12Imino acid content: 33%Substantially 100% of amino acids form the Gly-X-Y repeat structure.The amino acid sequence of CBE3 does not contain any of serine,threonine, asparagine, tyrosine, and cysteine.CBE3 has the ERGD (SEQ ID NO: 11) sequence.Isoelectric point: 9.34Amino acid sequence (SEQ ID NO: 1 in the Sequence Listing) (This aminoacid sequence corresponds to the amino acid sequence shown in SEQ ID NO:3 in WO2008/103041. Note that “X” at the end was modified to “P.”)

GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G

In the Examples described below, the above CBE3 was used as therecombinant gelatin (which is referred to as R-Gel), unless otherwisespecified.

Example 1 (1) Preparation of Recombinant Gelatin Gel

3% glutaraldehyde was added to a 10% recombinant gelatin aqueoussolution to an amount of 1/10 of the total amount. The finalconcentration of glutaraldehyde was 0.3%. A mixed solution obtained bystirring the thus prepared solution was poured into a silicon frame (30mm long×30 mm wide×2 mm high), and it was then left at rest at roomtemperature for 2 hours. Subsequently, the resultant was left at rest at4° C. for 12 hours, so as to obtain a chemically cross-linked gelatinsheet. This gelatin sheet was immersed in an excessive amount of glycineaqueous solution for 1 hour, and unreacted glutaraldehyde or aldehydegroups were inactivated. Subsequently, the resultant was washed withdistilled water twice and was then freeze-dried, so as to obtain across-linked recombinant gelatin gel. The freeze-dried recombinantgelatin gel was subjected to a crusher, New Power Mill (Osaka ChemicalCo., LTD.), so as to obtain powders. The obtained powders weresterilized with ethylene oxide gas before being used for samples inanimal experiments.

(2) Test Regarding Induction of Bone Regeneration in Rat Parietal BoneDefect Portions

Ten SD rats (male, 10- to 12-week-old, 0.3 to 0.5 kg) were used asexperimental animals. 0.8 ml/kg pentobarbital (Nembutal (registeredtrademark), Dainippon Sumitomo Pharma Co., Ltd.) was administered intothe abdominal cavity of each rat to anesthetize them. The parietal boneof each rat was exposed, and a circular bone defect portion having adiameter of 5 mm was created. The thus created bone defect portion wasfilled with approximately 10 mg of the sterilized, recombinant gelatingel, and the skin was then sutured.

Experiment Standard Groups:

Group 1: Only defectGroup 2: Recombinant gelatin gel (approximately 10 mg)Group 3: Pig-derived gelatin (high grade gelatin, TYPE: APAT, NippiInc.) (approximately 10 mg)Group 4: Collagen (Teruplug (trade name) crushed product, Olympus TerumoBiomaterials Corp.) (approximately 10 mg)Group 5: Bovine cancellous bone (Bio-Oss (trade name), Osteohealth)(approximately 20 mg)

On the 4^(th) week after performing the surgery, the rats weresacrificed by exsanguination under anesthesia with pentobarbital, andthe heads thereof were extirpated. The parietal bone including theembedded portions was subjected to histological observation with HEstaining.

(4) Results

The observation results of Groups 1 to 5 are shown in FIGS. 1 to 5. Inthe case of using the pig-derived gelatin and the recombinant gelatin,significant induction of bone formation was observed (FIG. 2 and FIG.3). Bone formation was not promoted by the collagen or a single use ofthe insoluble substrate (FIG. 4 and FIG. 5). However, using therecombinant gelatin of the present invention containing a large amountof RGD motif, bone formation was observed in a rat parietal bone defectportion by a single use of a bone supplementation material, which couldnot have formed bones so far (FIG. 2). This result demonstrated thateffective bone formation can be carried out without using aphysiologically active substance such as BMP that may cause sideeffects.

Example 2 Preparation of Recombinant Gelatin (R-Gel) and Animal GelatinFormation

An aqueous solution (pH 6) containing R-Gel or an animal gelatin (APAT,manufactured by Nippi Inc., concentration: 7.5%) and glutaraldehyde (GA,0.3%) was intensively stirred, and the solution was stirred for 3minutes while forming air bubbles. The obtained solution containing aninfinite number of air bubbles was left at rest at 4° C. overnight, soas to obtain a gelatinous solid containing a large number of airbubbles. The obtained gel was washed with a 0.1 M glycine aqueoussolution twice, unreacted glycine was inactivated, and the resultant wasthen washed with water four times. The gel that had been washed withwater was freeze-dried to obtain a spongy R-Gel and gelatin gel. Thissponge was crushed with a mill, so as to obtain spongy granules having agranular size of approximately several hundreds of μm. The internalstructures of the granules and Teruplug were observed under a scanningelectron microscope. As a result, it was found that they had the sameporous structure (FIG. 6).

An ethylene oxide-sterilized product of the spongy granules was used inthe following effectiveness evaluation. The R-Gel and animal gelatinthat had been cross-linked by the GA were each transplanted into rats ordogs, and the effectiveness thereof was then evaluated.

Example 3 Preparation of Rat Cranial Bone-Deficient Models

In order to evaluate the bone regeneration ability of R-Gel, rat cranialbone-deficient models used as a bone regeneration ability evaluationsystem were used (Tissue Eng (2007) 13(3): 501-12). The cranial bonefollows the same bone formation process (intramembranous ossification)as that of the alveolar bone. Thus, the cranial bone is generally usedin evaluation of dental bone supplementation agents.

Sprague-Dawley rats (SD rats, male, 10- to 12-week-old) wereanesthetized, and a circular defect portion (diameter=5 mm) was createdin the right-sided parietal bone using a drill (Osada Success 40, OsadaElectric Co., Ltd.) (FIG. 7). Bone fragments and blood existing in thedefect portion, which affected bone regeneration, were removed bywashing them with a normal saline, and the skin of the affected area wasthen sutured. After a predetermined period of time (3 weeks, 1, 2 and 3months) had passed, the rats were sacrificed by laparotomy andexsanguination. The defect portion of each rat was observed by the nakedeye, and the head portion was fixed with formalin and decalcified.Thereafter, a section obtained by slicing a block embedded in paraffinwas stained with hematoxylin and eosin (H & E), so as to prepare aspecimen. The pathological specimen was observed under an opticalmicroscope, and the ratio of a new bone to the defect portion wasdefined as a bone regeneration percentage (FIG. 8). Herein, the boneregeneration ability of the formulation was evaluated using such a boneregeneration percentage.

When the defect portion was observed, the defect portion of each rat wasslightly dished, and almost the entire defect portion was covered withsoft tissues. A small amount of white solid portion that seemed to be anew bone (regenerate bone) was observed around the cut section. Whenthis specimen was observed under an optical microscope one month afterperforming the surgery, approximately 30% of the defect portion wascovered with thin granulation tissues, and bone regeneration fromexisting bones was observed in approximately 10% of the defect portion.The bone regeneration percentage increased as the time passed. However,the percentage of the regenerate bone was approximately 30% even threemonths after performing the surgery. The amount of the bone regeneratedwas matched with the data of the publication (bone regenerationpercentage for 3 months: approximately 30%) (Tissue Eng (2007) 13(3):501-12), and thus, it was considered that a model system could beconstructed.

Example 4 Evaluation of Bone Regeneration Percentage in Rat Cranial BoneDefect Portion

A R-Gel formulation, an animal gelatin formulation, Teruplug (OlympusTerumo Biomaterials Corp.) and Bio-Oss Cancellous (0.25 to 1 mm,Osteohealth) were each transplanted into the rat cranial bone defectportion created in Example 3. A collagen membrane (BioGide, Osteohealth)was placed on each of the R-Gel, animal gelatin and Bio-Oss Cancellouseach having a dosage form that was a granular agent, so as to preventthe release of granules from the affected area. A group, to the affectedarea of which nothing had been applied, was defined as a control.

The bone regeneration percentage of an affected area, to which theBio-Oss Cancellous had been applied, was the same level as that of thecontrol, and it remained about 30% three months after performing thesurgery. In the case of the Teruplug that was a collagen sponge, a bonewas regenerated over time, and approximately 60% of the affected areawas repaired with the regenerate bone three months after performing thesurgery (FIG. 9). On the other hand, in the case of the R-Gel and theanimal gelatin, bone regeneration progressed at the same level as thatof the Teruplug until one month after the transplantation, butthereafter, their bone regeneration levels became higher than that ofthe Teruplug, and approximately 90% of the affected area was repairedwith the regenerate bone three months after the transplantation (whereinthe bone regeneration percentage was 1.5 times higher than that in thecase of the Teruplug). Accordingly, it can be said that the R-Gel andthe animal gelatin spongy granules have bone regeneration ability thatis higher than that of the Teruplug.

Example 5 Analysis of Pathological Specimens Using Animal Gelatin andR-Gel

In order to analyze bone regeneration using an animal gelatin (gelatin)and a recombinant gelatin (R-Gel) more in detail, the forms of tissuesaround each base material and the tissues of a regenerate bone wereanalyzed (FIG. 10). One month after the transplantation, a large numberof osteoblasts were found on the surfaces of both the R-Gel and gelatinbase materials, and new bones were found. Thus, no significantdifference was found between the tissue images of the two basematerials.

Two months after the transplantation, the presence of a large number ofosteoblasts, decomposition of the R-Gel by these cells, and formation ofa new bone with a few defects were observed in voids formed between theR-Gel and the new bone. On the other hand, at the interface betweenanimal gelatin and a new bone, a large number of fibroblasts, formationof fibrous soft tissues and a new bone containing a large number ofcicatricial fibrous tissues were observed. That is, the R-Gelregenerated a bone that was more dense than that regenerated by theanimal gelatin.

Example 6 Preparation of Dog Socket Preservation Model

A model was prepared according to the previous report (Araujo M et al.Int. J. Periodontics Restorative Dent. 28, 123-135, 2008.). Underanesthesia, the oral cavity of a beagle dog was disinfected with anIsodine solution, and the center between the second premolar (P2) andthe third premolar (P3) in both mandibles was excised with a fissurebur. A periodontal ligament around teeth in the distal position wasexcised with a knife, and the teeth in the distal position wereextracted using dental forceps and an elevator. The tooth extractionportion (wound socket after tooth extraction) was filled with a tamponthat had been immersed in a 10-fold diluted solution of an adrenalineinjection solution (Bosmin injection 1 mg/mL, Daiichi Sankyo Co., Ltd.)for hemostasis. The dental pulp of the remaining teeth in the proximalposition was removed using a reamer, the pulp cavity was then filledwith Gutta-percha, and it was then sealed with a pulp canal sealer.

Example 7 Evaluation of Effectiveness in Dog Model

The tooth extraction portion created in Example 6 was filled with BioOssCancellous (0.25 to 1 mm, Osteohealth), Teruplug (Olympus TerumoBiomaterials Corp.), R-Gel, or an animal gelatin, up to the crest of thealveolar bone. A collagen membrane (Biogide, Osteohealth) was placedbetween the infills other than the Teruplug and the gingiva. In the caseof a control, such an infill and a membrane were not placed.

The thickness of the gingiva in the tooth extraction portion wasexamined two months after the filling of the tooth extraction portion(FIG. 11). As a result, the thickness of the gingiva was decreased inthe case of an untreated control. The thickness of the gingiva wasmaintained in the portion into which the BioOss Cancellous had beentransplanted. The thickness of the gingiva was slightly decreased in theportion into which the Teruplug had been transplanted. In contrast, inthe portion into which the R-Gel had been transplanted, the thickness ofthe gingiva was maintained at the same level as that of the BioOss. Thatis to say, R-Gel exhibited effectiveness higher than that of Teruplug.

1-15. (canceled)
 16. A method for inducing granulation tissueregeneration which comprises administering a gelatin having the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 13, by itself, to a subjectat a site in need of granulation tissue regeneration.
 17. The method ofclaim 16, wherein the gelatin is crosslinked.
 18. The method of claim17, wherein the crosslinking is carried out using an aldehyde,condensing agent or enzyme.
 19. A method for supplementing granulationtissue which comprises administering a gelatin having the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 13, by itself, to a subject at asite in need of granulation tissue supplementation.
 20. The method ofclaim 16, wherein the subject has a granulation tissue defect.
 21. Themethod of claim 20, wherein the granulation tissue defect is caused byoral surgery, osteoporosis, or arthropathy.
 22. The method of claim 16,wherein the gelatin is in granule form.
 23. The method of claim 19,wherein the gelatin is in granule form.